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Abstract

Background

Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell
by receptor-mediated endocytosis. Several primary and low affinity co-receptors for
this flavivirus have been identified. However, the presence of these binding molecules
on the cell surface does not necessarily render the cell susceptible to infection.
Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection
and in designing control strategies.

Results

(1) Overlay protein binding assay showed two proteins with molecular masses of 80
and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited
cell binding and infection. (3) Both proteins were bound by all four serotypes of
dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified
by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled
surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against
Ae. aegypti midgut.

Conclusion

Our results strongly suggest that R67 and R80 are receptors for the four serotypes
of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.

Background

Dengue (DEN) is distributed worldwide in tropical and subtropical countries including
Mexico and the USA and is the most common vector-borne viral disease in humans. Infection
ranges from asymptomatic or mild self-limited illness (dengue fever, DF) to a severe
disease with spontaneous hemorrhaging (dengue hemorrhagic fever, DHF), or, most seriously,
to DEN shock syndrome (DSS) characterized by circulatory failure. Fifty million DEN
infections with 500,000 cases of DHF and 12,000 deaths occur each year [1]. In the years 2002 to 2004, 23,826 cases of DEN and 5,557 of DHF were reported in
Mexico [2].

The Dengue virus (family Flaviviridae, genus Flavivirus, species Dengue virus) infects mammalian [3] and vector cells. Neutralizing antibodies facilitate the binding and penetration
of DENV into human macrophages; the virus-antibody complex binds Fc receptors [4]. This mechanism does not explain virus entry in primary infections or in cells with
non-Fc receptors. Transmission electron microscopy shows that DEN viruses must attach
to the cells [5,6], suggesting that the cells must have virus receptor(s) on their surfaces to be susceptible
to infection. Proteins [7-12], heparan sulfates [13], LPS/CD14-associated binding proteins [14] and other glycoproteins [15] have been proposed as cellular receptors for DENV. In addition, DC-SIGN [16-18] has been suggested as a mediator of DENV infection in dendritic cells and could participate
in binding large numbers of other viruses such as HIV-1, Ebola and CytoMV to host
cell surfaces [19].

In C6/36 cells, two major polypeptides have been described with apparent molecular
weights of 67 and 80 kDa, which bind to DEN virus serotype 2 (DENV2) [9]. The mechanism by which the virus enters mosquito cells is unknown. The presence
of receptor(s) on the surface does not necessarily render a cell susceptible to infection.
In natural infection, DENV is first deposited in the mosquito vector and then in a
human host bitten by the vector during a blood meal. Therefore, it is necessary to
study receptors in the mosquito midgut (MG) to determine which binding proteins serve
as true virus receptors; they may be relevant to treating DEN infection and in designing
control strategies. This is especially important because mosquitoes differ in their
susceptibilities to infection, resulting in different vector competences [20-22].

The present study shows that the proteins R80 and R67 are the putative receptors in
the MG of Ae. aegypti. Moreover, proteins with the same apparent molecular weights were purified by DENV2
affinity chromatography from the MG of this mosquito and from C6/36 cells. In addition,
a protein with molecular weight of 57 kDa was also purified from mosquito MG extracts
by affinity chromatography. Immunoprecipitation of radiolabeled membranes using Ae. aegypti anti-MG antibodies showed that R80 and R67 are contained in mosquito cell membranes.
Furthermore, the four serotypes of DENV recognized the same proteins on Ae. aegypti and C6/36 cells.

Results

Identification and purification of the putative DENV2 receptors in Ae. aegypti midgut
by the virus overlay protein binding assay

Mosquito MG proteins were extracted with 0.05% of Triton X-100, separated by SDS-PAGE,
blotted on to a PVDF membrane and incubated with biotinylated DENV2 virus to identify
the putative receptors. Two prominent polypeptides with apparent molecular masses
of 67 and 80 kDa were observed (Figure 1, panel A, lane 1). The negative control without virus showed no bands (data not shown).

Figure 1.VOPBA and DENV2 affinity chromatography of the virus dengue receptors. Proteins from Aedes aegypti MGs were extracted with 0.05% Triton X-100, separated by 10% SDS-PAGE and blotted
on to a PVDF membrane. The putative receptors were revealed after incubation with
DEN2 virus and peroxidase-labeled goat anti-mouse antibody (panel A, lane 1) as described
in the methods section. Receptors were purified from C6/36 cell membranes (panel A,
lane 3) or MG (fractions 1 and 2, panel B) by DENV2 affinity chromatography using
a DEN2-Sepharose™ 4B column as described in the methods section. The apparent molecular
weights of these proteins are shown on the right side of panel A and on the left side
of panel B. Proteins separated by SDS-PAGE were silver stained. The protein pattern
of total C6/36 membranes is shown in panel A lane 2. Molecular weight markers are
shown on the left side in panel A and on the right side in panel B.

Immunoprecipitation of the putative receptors from C6/36 cell apical surfaces

To confirm that the putative receptors in Ae. aegypti MG epithelial cells are similar to those in Ae. albopictus C6/36 cells, the apical surfaces of C6/36 cells were labeled with 125I and precipitated with anti-C6/36 cell membrane and anti-MG antibodies. Antibody
specificity was tested by immunoblotting (Figure 2, panel A, lanes 2, 4). Proteins with apparent molecular weights over 180 kDa were
recognized. The negative control with pre-immune serum showed no bands (Figure 2, lanes 3 and 5).

Figure 3.Immunoprecipitation of labeled membrane proteins. Proteins from C6/36 cell apical surfaces radiolabeled with 125I (lane 1) were immunoprecipitated with polyclonal anti-MG (lane 2) or anti-membrane
(lane 3 and 4) and separated by 10% SDS-PAGE. The molecular weights of the immunoprecipitated
proteins are shown on the left side. A negative control using an unrelated antibody
(anti-actin) is shown in lane 5. Labeled apical cell surface proteins recognized by
DEN1 (lane 7), DEN2 (lane 8), DEN3 (lane 9) and DEN4 (lane 10) were immunoprecipitated
with specific anti-DENV as described in the methods section. A control without DEN
virus was included (lane 6). 125I-labeled proteins immunoprecipitated by anti-EP were used as a positive control (lane
11). All proteins were separated by 10% SDS-PAGE and labeled proteins were detected
by autoradiography. The molecular weights of these proteins are shown on the right
side. Molecular weight markers are shown on the left side.

Apical cell surface proteins bound the four serotypes of DEN virus

To determine whether different serotypes of DENV recognized the same receptors, labeled
apical cell surface proteins from C6/36 cells were incubated with DENV1, DENV2, DENV3
or DENV4 (Figure 3 lanes 7–10, respectively) and precipitated by incubation with antibodies against
each DENV serotype. All four serotypes led to the immunoprecipitation of proteins
with molecular masses of 80 and 67 kDa (Figure 3, lanes 7–10). Labeled C6/36 apical membrane proteins were precipitated by anti-EP
antibodies as a positive control (Figure 3, lane 11). The control without DEN virus showed no polypeptides (Figure 3, lane 6).

Figure 4.Immunoblotting of total C6/36 extracts. Extracts from C6/36 cells treated as in Figure 2 were tested with pre-immune mouse
serum (lane 1), anti-EP (lane 2), anti-R80 (lane 3) or anti-R67 (lane 4). Molecular
weight markers are shown on the left side and the proteins detected by the antibodies
on the right side.

There was no immunofluorescence staining when C6/36 cells were incubated with pre-immune
serum (Figure 5A). In contrast, anti-membrane, anti-MG, anti-EP, anti-R67 and anti-R80 antibodies
all stained the C6/36 membranes (Figure 5B–F, respectively). Anti-R80 also stained the cytoplasm (Figure 5F).

In order to test antibody blocking of DENV binding, C6/36 cell monolayers were incubated
with anti-EP, anti-R67, anti-R80 or anti-MG antibodies or pre-immune serum, then with
125I- DEN2 virus as described in the methods section (Figure 6). Anti-EP, anti-R67 and anti-R80 decreased virus binding by up to 40%. Binding was
inhibited by 70% when anti-MG was diluted 1:10 (Figure 6).

Figure 6.Blocking of DEN-2 virus binding by specific antibodies. C6/36 cell monolayers were incubated with 1:10 or 1:1000 diluted pre-immune serum
(PI), anti-EP, anti-R67, anti-R80 or anti-MG. To assay the binding of radiolabeled
virus, cells were dissolved in 3% SDS in all experiments. The total [125I]DENV2 virus bound was 5,286 ± 300 cpm. The experiments were repeated at least 3
times in quadruplicate.

To test the inhibition of DENV2 infection by the specific anti-receptor antibodies,
C6/36 cell monolayers were incubated separately for 1 h in media containing pre-immune
serum, anti-EP, anti-R67 or anti-R80 diluted 1:10 or 1:50, before being infected with
DENV2. Infection was allowed to proceed for 8 days and the amount of infectious virus
was determined by viral plaque assays in LLC-MK2 cells (Figure 7). Maximal inhibition of infection was obtained with anti-EP and anti-R67 diluted
1:10 (Figure 7). These antibodies inhibited DEN 2 replication in the C6/36 cells nearly 1000-fold.
Anti-R80 showed a relatively minor inhibitory effect of approximately 100-fold. Anti-EP,
anti-R67 or anti-R80 diluted 1:50, and pre-immune serum, did not inhibit DEN2 replication
in these cells.

Figure 7.Inhibition of DENV2 infection by specific antibodies. C6/36 cell monolayers were incubated separately in the presence of pre-immune serum,
anti-EP, anti-R67 or anti-R80 at 28°C for 60 min and then DENV serotype 2 was added
(600 PFU/well) and incubated for 30 min at 4°C. After washing, fresh culture medium
was added and the cells were incubated for 8 days. Pre-immune serum and antibodies
were diluted 1:10 and 1:50. Viral titers in the supernatants were determined by viral
plaque assay in LLC-MK2 cells. The experiments were repeated at least 3 times in quadruplicate.

Discussion

When a mosquito takes a viremic blood meal, virions interact with receptors on the
midgut epithelial cells and penetrate and infect them. Arbovirus blocked at early
stages of midgut infection is considered a midgut infection barrier (MIB). A midgut
escape barrier (MEB) is considered when infectious virions do not disseminate to hemoceles,
or disseminate but do not infect secondary target organs.

Most studies of flavivirus vector competence in Ae. aegypti indicate that MIB is a major determinant of transmission [23-25] and shows wide variation both among and within Ae. aegypti populations for flaviviruses including DENV [20,22,25]. Moreover, Ae. aegypti MG is generally considered the best candidate tissue for disrupting the virus life
cycle within the mosquito because it is the earliest interface between insect and
virus (see above). This strongly suggests that DENV attachment to MG epithelial cell
receptors is critical for understanding the initial virus-vector interactions and
will help to explain MIBs to DENV infection and variations in vector competence. Furthermore,
we would expect that virus serotype and genotype would influence virus attachment
to midgut receptors.

Thus, identification of viral receptors in the MG represents a critical step in understanding
vector competence and designing possible targets for preventing viral entry to cells
and therefore inhibiting the infection. Feasible approaches to intervention are monoclonal
antibodies blocking the receptor, and synthetic peptides mimicking the viral receptor
and thus competing with the host receptor for virus attachment. This will allow novel
strategies for the control and prevention of DEN to be developed. Published data have
shown that the viral E protein is involved in target cell recognition [6,26,27]. Recent structural and genetic evidence [28,29] suggests that the prM/M stem-anchor region is also likely to play a role in virion
entry to cells. Purification by DENV 2 affinity chromatography using native virus
is a novel approach to obtaining the putative protein receptors for DENV.

Several authors using the overlay assay have identified polypeptides with molecular
masses of 27, 45, 67 and 87 in macrophages [8], 40 and 70 in myelomonocytes [30], 45 and 72 kDa in B and T cells from humans [31], and two heparan sulfate containing cell-surface binding proteins resolving at 19
and 37 kDa in hepatocytes [32]. The data in the present paper strongly support the view that the 67 and 80 kDa proteins
are receptors for DENV 1, 2, 3 and 4 in MG cells from Aedes aegypti. Furthermore, we have purified these proteins for the first time from both Ae. albopictus C6/36 cells and Ae. aegypti MGs using DENV2 affinity chromatography; we also generated specific antibodies against
them, which inhibited binding and infection of cultured cells by DENV 2. These results
contrast with those of Yazi-Mendoza et al. [33], who showed that DENV 4 binds to a 45 kDa protein in mosquito tissues. The results
in Figure 2 clearly demonstrate that DENV 4 recognizes R67 and R80 in C6/36 cell membranes. A
plausible explanation is that Yazi-Mendoza et al. [33] used total cell extracts for their VOPBA, while our results were obtained from plasma
membranes, although viruses are known to have several receptors [7]. In any event, further experiments are needed to settle the point. Interestingly,
Jindadamrongwech & Smith [7] found a serotype-specific heterogeneity among DENV binding proteins on HepG2 human
Liver cells. It is therefore possible that different cell lines have different specificities
for virus serotypes.

The specific antibodies against R67 and R80 inhibited radiolabeled DEN virus binding
and the highest inhibition was observed with anti-MG antibodies, suggesting that other
cell membrane molecules may participate in virus binding. Candidates include non-sialic
acid carbohydrates, since we previously showed that sialic acid does not participate
in virus binding [9]. In agreement with our results, Zieler et al. [29] failed to detect sialic acid in Ae. aegypti MG. More recently, Thaisomboonsuk et al. [34] reported the inability of neuroaminidase to inhibit DEN-2 virus binding to insect
cells.

As we mentioned earlier, DC-SIGN has also been reported as a functional receptor on
human dendritic cells [15,16] for DENV and other flaviviruses. Further studies are needed to determine the involvement
of DC-SIGN as receptor in DENV infection in Ae. aegypti mosquitoes.

Collectively, the results corroborate the notion that DENV utilizes multiple cell
surface molecules for binding to and infection of target cells, some of which may
be common to all cells and shared among several viruses [6]. Interestingly, Sakoonwtanyoo et al. [12] suggested that one of these protein receptors for DENV 2, 3 and 4 may be a laminin-binding
protein with an apparent mass of 50 kDa. Although we failed to detect a protein with
this molecular mass, this does not eliminate the possibility.

In summary, specific membrane molecules are required for DENV binding and the nature
of these molecules seems to depend on cell type. Interestingly, our results show that
R67 and R80 are the putative receptors in the MG of Ae. aegypti mosquitoes, the principal DENV vector in America, and in Ae. albopictus C6/36 cells. In addition, these receptors are specific for all four serotypes of DENV.

Conclusion

This paper documents for the first time that the proteins R67 and R80 are putative
receptors for dengue virus in the midgut of Ae. aegypti and in Ae. albopictus C6/36 cells, since specific antibodies against these proteins inhibited the binding
and cytopathic effects of the virus. Furthermore, we have shown that these receptors
are specific for all four serotypes of DENV.

Methods

Mosquito culture

A. aegypti mosquitoes collected as larvae in Monterrey, Mexico were laboratory-reared and maintained
at 28 ± 2°C and 80% RH under a 12:12 h (L:D) photoperiod using standard mosquito-rearing
procedures [35]. After day 4, adult MGs were dissected and stored at -80°C until use.

DENV infection cells

Ae. albopictus clone C6/36 cells were grown at 28°C as previously described [36]. After 18 h of culture, the cells (2 × 106/100 mm plate) were infected with 0.2 ml DENV2 inoculum with an input MOI of 600 PFU
per plate and incubated at 28°C for 10 days.

Virus passage in Vero cells was clarified from cell culture supernatants. Viruses
were concentrated and purified as described by Putnak et al. [37]. Titers of virus stocks made in LLC-MK2 cells [38] were 8 × 108 PFU/ml for each DENV strain. Control antigens harvested from uninfected Vero cells
were prepared in the same manner.

The purified viruses were iodinated using 1 mCi of 125I (Amersham Pharmacia Biotech) as described elsewhere [39]. The specific activity was 4.7 × 109 cpm/mg of protein.

Biotinylation was performed as described previously [9]. The viral pellet was stored at -70°C until use.

Membrane preparation

C6/36 cell membranes were prepared essentially as described elsewhere [40] by scraping the cells from confluent plates in the presence of PBS. This procedure
has been described in detail for C6/36 cells [9]. Labeled membrane proteins were identified at the 20% interface of the gradient and
verified by SDS-PAGE.

DENV2 affinity chromatography

DEN2 viruses (8 × 108 PFU/ml) were bound covalently to 1 g of CNBr-activated Sepharose™ 4B as recommended
by the manufacturer (Amersham Biosciences). The DENV2-Sepharose™ column was stored
in 0.002% sodium azide at 4°C until use.

MG protein extract (2 mg), or 100 μg C6/36 cell membranes [4,9], was applied to a DENV2-Sepharose™ 4B column (1 ml) equilibrated in buffer E and
washed with the same buffer. The DENV2-binding proteins were eluted with buffer E
containing 0.5 M NaCl. Fractions of 0.5 ml were collected, and the protein content
was monitored by the Bradford method [41] and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate on 10% gels [42]. The eluted proteins (EP) were stored at -70°C.

Radiolabeling of C6/36 cell membrane proteins

Proteins from the apical surfaces of confluent monolayers were iodinated by the lactoperoxidase
method [39]. Labeled membrane proteins (specific activity 1.4 × 106cpm/mg) in buffer E were used immediately or stored at -70°C.

Antibodies

To obtain specific anti-R80 and anti-R67, proteins retained by the DENV2-Sepharose™
4B column were separated by 10% SDS-PAGE. After silver staining, each of the two main
protein species (R80, R67) was excised from the gel, cut in small pieces, suspended
in PBS and mixed with an equal volume of Titer-Max adjuvant (CyTRx Corporation) to
immunize two groups of BALB/c mice. Pre-immune sera were obtained before immunization.
Proteins eluted (EP) from the DENV2 affinity column were used to obtain specific polyclonal
antibodies. A total of 10 μg of this protein was used to immunize the mice. MG extracts
(100 μg), or C6/36 cell membrane extracts (50 μg), were also used to immunize mice
[9]. After fifteen days, the mice received a booster; they were bled after thirty days.
Sera were stored at -70°C until use. Antibody specificity was tested by ELISA and
immunofluorescence. Negative controls using pre-immune sera were included in all assays.

Binding and infection blocking assays

C6/36 cells (1.5 × 104) were cultured overnight in 96-well plates as described above. For binding assays,
the cells were incubated for 60 min at 4°C with pre-immune serum, anti-EP, anti-R80,
anti-R67 or anti-MG diluted 1:10 or 1:1000 in serum-free medium. Other cells were
left untreated. 125I-DENV of each serotype was added (600 PFU/well) and incubated for 30 min at 4°C.
After washing to remove non-bound labeled virus, cells containing the bound virus
were solubilized in 3% SDS and the radioactivity was counted. For infection assays,
cells were incubated for 60 min with pre-immune serum, anti-EP, anti-R80 or anti-R67
diluted 1:10 or 1:50 at 28°C and then DENV serotype 2 was added (600 PFU/well) followed
by incubation for 30 min at 4°C. After washing, fresh culture medium was added and
the cells were incubated for 8 days. The viral titer was determined in each supernatant
by viral plaque assay in LLC-MK2 cells [43]. Four wells were assessed for each experimental condition.

Immunofluorescence

C6/36 cell monolayers were stained as previously described [9]. Pre-immune sera as well as each of the polyclonal antibodies (anti-R80, anti-R67
and anti-MG) were diluted 1:100 and incubated overnight at 4°C. After washing with
PBS, the cells were incubated with fluorescein-conjugated goat anti-mouse (1:500)
antibodies (Hyclone Laboratories, Inc., Utah). After further washing, they were mounted
in 50% glycerol and observed by fluorescence microscopy.

Electrophoretic blotting

Proteins were electrophoresed in 10% SDS-PAGE [42] and transferred to nitrocellulose paper [44]. Membranes were incubated with anti-EP, anti-R80, anti-R67 or anti-MG diluted 1:50.
The goat anti/mouse IgG second antibody was conjugated to alkaline phosphatase and
color development was measured as recommended by the manufacturer (Zymed Laboratories).

Virus overlay protein binding assay (VOPBA)

Mosquito MG proteins were separated by 10% SDS-PAGE as described above and blotted
on to PVDF membranes (BioRad) by Towbin's technique [44]. The procedure was followed as previously described [9] with some modifications. Briefly, the electrophoretic blots were incubated overnight
with unlabeled virus, washed with PBS and incubated overnight with monoclonal anti-flavivirus
(diluted 1:100) at 4°C. After washing with PBS, they were incubated for 2 h at room
temperature with peroxidase-labeled goat anti-mouse (Kirkegaarde Perry Laboratories,
USA) diluted 1:1000 in 5% skimmed milk in PBS. Finally, they were washed with PBS,
and reactive proteins were visualized by developing with the chromogenic substrate
4-chloro-1-naphthol/hydrogen peroxide. The reaction was stopped after 1 h by washing
with water.

Receptor-virus immunoprecipitation

Radiolabeled cell membrane proteins (5 μg) solubilized in buffer E containing protease
inhibitors (see above) were incubated for 2 h at 37°C with 10 μl of each DENV serotype
(6.0 × 106 PFU/ml) and anti-DENV 1, 2, 3 or 4 antibodies diluted 1:100. The mixture was centrifuged
at 5,000 × g for 15 min and washed twice with PBS. The supernatant was discarded and
the proteins from the pellet were suspended in SDS gel-loading buffer and separated
by 10% SDS-PAGE. Labeled polypeptides were detected by autoradiography [45]. Negative controls were included by incubating the same preparations with anti-actin,
an unrelated antibody.

Authors' contributions

RFMC carried out the virus purification experiments, DENV infection of the cells,
affinity purification of DENV2 MG receptor, electrophoretic blotting and the virus
overlay protein binding assay. HAEA purified the receptors from C6/36 cell membranes,
and carried out the binding and infection blocking assays and immunofluorescence studies.
RT radiolabeled the membrane proteins and obtained the membrane preparations, raised
antibodies and performed the receptor-virus immunoprecipitation. ADB cultured and
field collected mosquitoes. MCN assembled the manuscript and participated in data
analysis. MLM proof-read and assembled the manuscript. All authors read and approved
the final manuscript.

Acknowledgements

We acknowledge the M.Sc. Gustavo Limón Camacho for the standardization of the protein
receptor purification assay. This research was supported by United States Public Health
Service Grant AI 45430 subgrant G-46321.